Clostridium difficile causes severe diarrheal disease that results in billions of dollars per year in increased health care costs and more than 20,000 deaths annually in the United States. In the past decade, an epidemic strain has emerged (NAP1/B1/ribotype 027, toxinotype III) that is associated with a significant increase in morbidity and mortality. The incidence of C. difficile 027 epidemic infections has increased at an alarming rate in the past 10 years and these infections have spread globally. Infections caused by this strain have lower cure rates and higher rates of relapsing disease than infections caused by other C. difficile isolates. Despite the predominance of 027 epidemic isolates, we do not know what properties of this lineage have enabled the rapid ascent of this strain. The long-term goal of this project is to determine how epidemic isolates of C. difficile proliferate in the host o that new approaches for preventing and treating infections can be identified. Based on our data, we hypothesize that epidemic C. difficile are more pervasive because they more successfully survive the effects of antimicrobial peptides (AMPs), allowing them to colonize and replicate better in the intestine. The specific objective of this application is to identify the genetic mechanisms in 027 epidemic C. difficile that confer increased AMP resistance in the host. Capitalizing on our previous experience with C. difficile antimicrobial peptide resistance, we will meet this objective through the experiments detailed in two specific aims. First, we will reveal the genetic mechanisms of AMP resistance in epidemic C. difficile strains through genetic analysis of mutants defective in AMP resistance. Next, we will determine the contribution of AMP resistance mechanisms to colonization and infection by epidemic isolates. The expected contribution of the proposed research is the identification of genetic mechanisms in 027 epidemic strains of C. difficile that confer increased antimicrobial peptide resistance compared to non-epidemic strains. This contribution is significant because it is the first step in understanding how the increased antimicrobial peptide resistance of 027 epidemic strains influences the spread of this important pathogen.